Serial femtosecond crystallography (SFX) is a novel structural biology technique that allows challenging protein structures to be solved from sub-micrometer/micrometer sized crystals at room temperature (Chapman et al., 2011). In SFX, nanocrystals and/or microcrystals are delivered in a liquid (DePonte et al., 2008) or a viscous stream (Weierstall et al., 2014) into the beam path of a hard X-ray free-electron laser (XFEL). XFEL radiation is composed of femtosecond pulses typically delivered at a rate of 1-120 Hz, and diffraction patterns are obtained before the crystals are destroyed (Neutze et al., 2004; Barty et al., 2012). SFX currently requires large data sets because the diffraction patterns are acquired from individual randomly oriented protein crystals. Most SFX experiments thus far have been based on protein crystals delivered using a gas dynamic virtual nozzle (GDVN), where the crystals are delivered to the X-ray beam in their mother liquor (DePonte et al., 2008).
The gas-focused GDVN liquid jet moves at a velocity of 10-20 m s−1, which delivers crystals much faster than required to replenish the protein crystals between X-ray pulses at a pulse repetition rate of 120 Hz. Therefore, approximately only one out of every 10,000 crystals is probed by the X-ray pulses (Weierstall et al., 2014). This type of liquid jet can consume 10-100 mg of protein for the collection of a complete data set, which is particularly problematic for membrane proteins and other proteins that can only be produced in small amounts.
Membrane proteins are an important class of proteins that are of very high relevance in biology, compromising 60% of all current drug targets (Hopkins & Groom, 2002). However, structure determination of membrane proteins lags far behind soluble protein structure determination, with less than 550 unique membrane-protein structures determined so far out of over 100,000 structures currently deposited in the Protein Date Bank.
Membrane proteins are insoluble in water and therefore have to be extracted from the membrane in the form of protein-detergent micelles. Most membrane-protein structures are obtained by either crystallization in solution in the form of protein-detergent micelles or crystallization in the lipidic environment of the lipidic cubic phase (LCP), a method for membrane-protein crystallization pioneered by Landau & Rosenbusch (1996).
LCP is a liquid crystalline phase that is spontaneously formed upon mixing monoacylglycerols (MAGs) and water, producing a continuous three-dimensional network of curved bilayers arranged into a cubic lattice with two networks of interconnecting continuous aqueous channels (Caffrey, 2015). The architecture of the lipid formation encourages type 1 crystal packing and has similar properties to the native cell membrane (Caffrey, 2015). Crystallization in LCP has been successful for structure determination of a wide range of membrane proteins, including microbial rhodopsins, photosynthetic complexes, β-barrels, enzymes, transporters, ion channels and especially G-protein-coupled receptors (GPCRs), a class of membrane proteins with high medicinal impact (Cherezov, 2011).
A new crystal-delivery system has been developed for SFX which allows the delivery of crystals grown in LCP to the XFEL beam (Weierstall et al., 2014). The high viscosity of LCP results in a much slower flow rate of the stream, thus drastically decreasing the net mass of protein needed for structure determination by SFX. LCP as a delivery medium has been successfully used to determine GPCR structures using an XFEL (Liu et al., 2013; Fenalti et al., 2015; Weierstall et al., 2014; Zhang et al., 2015). Crystallization of membrane proteins in LCP has been highly optimized, contributing to the structures of over 60 unique membrane proteins to date (Caffrey & Cherezov, 2009). However, it has been challenging to crystallize large multi-domain membrane complexes in LCP owing to the curvature associated with the lipid bilayer and the low diffusion constants of large membrane-protein complexes in LCP. To date, the majority of membrane-protein structures solved by X-ray crystallography have been determined from crystals of protein-detergent micelles grown in solution, which have also been successfully used for SFX experiments (Chapman et al., 2011; Aquila et al., 2012; Johansson et al., 2012, 2013; Kupitz et al., 2014). These membrane-protein crystals were delivered either with the GDVN liquid injector (DePonte et al., 2008), requiring large amounts of protein, the gel injector (lipidic cubic phase injector; Weierstall et al., 2014) or an electrospinning injector (Sierra et al., 2012), which uses less protein but uses high electric fields which could be problematic for crystal stability.
To date, all membrane-protein structures delivered in LCP for SFX (Weierstall et al., 2014; Liu et al., 2013; Fenalti et al., 2015; Zhang et al., 2015) have been based on crystals that were grown in LCP. Mixing of membrane-protein crystals grown in the form of a protein-detergent micelle with LCP typically leads to dissolution of the crystals, very likely caused by partitioning of the detergent, which forms the protein-detergent micelle, into the lipidic phase. This leads to depletion of the detergent in the protein-detergent micelles in the crystals, resulting in denaturation of the protein. Recently, two other viscous media, a mineral oil-based grease and petroleum jelly, have been described as alternative crystal-delivery carriers (Sugahara et al., 2015; Botha et al., 2015). The grease mixture (Sugahara et al., 2015) has been used to deliver crystals to the XFEL beam for SFX data collection of soluble model proteins at the SPring-8 Compact Free Electron Laser (SACLA XFEL), while petroleum jelly (Botha et al., 2015) has been used to deliver lysozyme at the Swiss Light Source (SLS). Both of these delivery methods have so far only been demonstrated at ambient pressure and they produce significant and undesirable Debye-Scherrer rings in the region of 3.77-5 Å. No evidence has been presented to date that show either medium to be suitable for the delivery of multi-protein complexes, membrane proteins, nucleic acids, macromolecular complexes, or viruses. Thus, there would be utility in an inert medium for the delivery of both soluble and membrane proteins, nucleic acids, macromolecular complexes, and viruses to the XFEL beam at slow flow rates.